The effort of our laboratory is currently directed toward three separate projects: (1) modeling human sarcomas in the mouse; (2) investigating the molecular genetic causes underlying a human neuropsychiatric disorder, the OCD-spectrum disorder trichotillomania; and (3) developing the technology to functionally interrogate, on a genome-wide basis, any mammalian genome. The motivation for addressing each project is unique to the project.
Mouse Models of Human Sarcomas
Because sarcomas are not as common as carcinomas, they have not received comparable attention from the research community. However, sarcomas predominantly affect a critical human population: children, adolescents, and young adults. Many sarcomas are extremely aggressive, and current therapies, including surgery, radiation, and chemotherapies, are often ineffectual. A prognosis of 50–80 percent mortality, five years subsequent to first presentation, is not uncommon. It is clear that future effective therapies will have to be directed to specific molecular targets driving each individual sarcoma. Development of such therapies will require an understanding of the molecular players and pathways responsible for initiation and progression of the malignancy. I believe that such knowledge can only be attained through the use of animal models that faithfully recapitulate the human cancer. Fortunately, the criterion for authenticity of the animal model is now stringent, genome-wide expression profiling, which provides a detailed fingerprint unique to each cancer. An authenticated model can be used as a platform to interrogate the mechanism of tumor progression as well as to develop effective therapies.
From an animal modeler's point of view, analyses of sarcomas commonly have enormous advantages compared to analyses of carcinomas. (1) A specific reciprocal interchromosomal translocation often initiates and is also required for maintenance of the sarcoma (i.e., it is a major driver of the malignancy). Carcinomas, on the other hand, typically employ many drivers, each fractionally contributing to the manifestation of the malignancy. For most human carcinomas the initiating events have not been defined, and it is not known whether independent tumors of the same class are even initiated by the same mechanisms. (2) Genome instability is not a hallmark of most sarcomas. Often the only observable karyotype abnormality present in the sarcoma is the chromosomal translocation itself that defines the sarcoma. These common features of sarcomas greatly simplify generating mouse models that closely recapitulate the human cancer, identifying the causative agents responsible for tumor progression, and conceptualizing meaningful therapies based on this knowledge.
To date we have developed and authenticated three mouse models of human sarcomas: alveolar rhabdomyosarcoma, synovial sarcoma, and clear-cell sarcoma. We are working on modeling a fourth, Ewing's sarcoma. These models are now being used by the research community at large and should significantly impact the future management of these devastating malignancies. Because of the lower frequency of these sarcomas relative to carcinomas, coordination between medical centers is often required to gather sufficient numbers of patients for the implementation of clinical trials. Under these circumstances, mechanistic insights derived from authenticated sarcoma mouse models should be used to prioritize clinical trial efficacy.
Molecular Genetic Causes of Neuropsychiatric Diseases
Our entry into modeling neuropsychiatric disorders arose serendipitously, a result of our long-standing interest in the role of Hox genes in mammalian development. Disruption of Hoxb8 in mice resulted in an unexpected phenotype, a behavioral deficit similar to the human obsessive-compulsive disorder (OCD) spectrum disorder, trichotillomania, the compulsive removal of body hair. We then demonstrated that the only cells in the brain that express Hoxb8 in mice were microglia, the immune cells of the brain. Furthermore, a transplant of normal bone marrow into lethally irradiated Hoxb8 mutant mice cured the mice of their behavioral disorder. Conversely, transplantation of Hoxb8 mutant bone marrow into irradiated normal mice conferred the behavioral deficit to the normal mice. That the behavior malady was caused by mutant bone marrow was also demonstrated by conditional mutagenesis that restricted the generation of the Hoxb8 mutation to definitive hematopoiesis using the Cre/loxP system. We concluded that Hoxb8 mutant microglia are causative in mice for the behavioral deficit that is similar to human trichotillomania. We have correlated the presence of a human DNA polymorphism in the Hoxb8 locus that tracks with human trichotillomania. This polymorphism is not within the DNA sequence that encodes Hoxb8 protein but could affect Hoxb8 mRNA splicing. We are transferring the human polymorphism into the mouse Hoxb8 locus to determine if this polymorphism reduces Hoxb8 protein production and by what mechanism.
The next question that arises is how microglia modulate the activity of the neural circuits that are responsible for controlling compulsive behavior. Correlations between neuropsychiatric disorders, such as monopolar depression, bipolar depression, schizophrenia, autism, OCD, OCD-spectrum disorders, and Alzheimer's disease, have been documented for multiple decades. However, cause and effect for these correlations were not determined. Is the patient depressed and the activity of their immune system affected, or do defects in their immune system increase the risk of acquiring depression? In our mouse model, defective microglia (the immune system) give rise to the specific behavioral disorder, compulsive pathological hair removal. Using optogenetics, we hope to determine how microglia modulate the appropriate neural circuits responsible for the aberrant behavior. The OCD circuit in humans has been defined by functional MRI (magnetic resonance imaging), so we know where to look in the brain for microglia-neuron interactions.
Prior to our study, microglia were viewed as a uniform population of cells. Our studies suggest the existence of at least two populations: a resident population that arises in the yolk sac as a product of primitive hematopoiesis (60 percent of total microglia) and Hoxb8-expressing microglia that arise later from definitive hematopoiesis (40 percent of total microglia). Since 60 percent of the microglia (the non-Hoxb8-expressing, resident microglia) cannot compensate for the Hoxb8 mutant microglia, it appears that with respect to the behavioral deficit, the two populations of microglia have separate functions. How similar, or dissimilar, these two populations are remains to be determined. The distribution of these two populations of microglia in the brain is different: the concentration of Hoxb8-expressing microglia is relatively higher in the regions of the brain previously identified as comprising the OCD circuit.
Genome-Wide Analysis by Chromosome Transfer
Currently molecular genetic analysis of animal species within our biosphere is restricted to a handful of model organisms that—with respect to size, fecundity, generation time, and husbandry—are readily accessible to a laboratory setting. Primary emphasis has been on the enormous similarities, at the molecular level, exhibited among these disparate species. However, there is likely to be as much learned from determining the molecular genetic causes that distinguish two species as there is from studying their similarities. For example, the gene content of all mammals is more than 99 percent similar. Yet the size, shape, and capabilities of mammals vary enormously. The same set of genes in a mouse, a whale, or a human are apparently regulated differently, but which genes are responsible for the marked differences? How complex is this process, and how might we approach analysis of such questions?
Because the cost of DNA sequencing continues to drop, the availability of fully annotated genomes will soon be extensive. In silico comparisons of these vast data sets are likely to reveal intriguing hypotheses pertinent to human health and productivity. For example, why do some mammalian species appear relatively impervious to the consequences of infection by a broad range of viruses—such as Ebola, Marburg, Hendra, Lyssa, and severe acute respiratory syndrome (SARS)—that are lethal to humans? Although in silico comparisons of many mammalian genomes may reveal intriguing hypotheses, such comparisons are not likely to permit tests of these hypotheses. Such tests will require functional analysis of the mammalian genome in question. We are proposing to develop a technology to allow functional analysis, at the genome-wide level, of any mammalian species, particularly those not readily accessible to molecular genetic interrogation.
We are developing a methodology for systematic evaluation of the genetic and epigenetic causes of the extensive differences between mice and another chosen mammalian species. This technology will use the mouse as a surrogate expression host for genes of another mammalian species on a genome-wide basis. To make this economically feasible, we envision generating a set of mouse lines (~120 mice), each containing a different chromosomal fragment. Collectively these mouse lines would represent almost the entire genome of the chosen species (i.e., more than 92 percent representation in the first round). This procedure, which we have termed mammalian genome-wide functional analysis by chromosome transfer (MGWFACT), would enable functional analysis of any mammalian genome using all of the tools currently available in the mouse.
To test this technology, we are analyzing Myotis lucifugus, the little brown bat. We have chosen the bat because of the enormous differences in shape and capabilities between it and the mouse. Despite these differences, these two species are similar with respect to size, heart rates, body temperature, and metabolic rates. Thus, with respect to physiology, we expect the two species to be compatible. Furthermore, the difference in capabilities is of particular interest to our laboratory and to human health and productivity. For example, bats are well known for their tolerance of infection by viruses that are lethal to humans. In the SARS epidemic of 2003, the virus was spread from bats to humans. Bats have been shown to be reservoirs for some of the world's most highly pathogenic viruses, including Ebola, Marburg, Hendra, and Lyssa. The mechanisms by which bats tolerate infection by such virulent pathogens are unknown. Many species of bats, including M. lucifugus, have lived at enormous densities during their evolution and have developed the biology to cope with living at such high densities. This may account for their marked resilience to viral infection. Studying this problem directly in bats is problematic since the majority of bat species, including M. lucifugus, cannot be maintained in a laboratory.
Another attractive feature of M. lucifugus that contrasts to Mus musculus is longevity. The little brown bat lives in the wild for more than 40 years; laboratory mice, under favorable husbandry conditions, live 2.0–2.5 years. This difference in life span cannot be explained by hibernation, since related bat species that do, or do not, hibernate have similar life spans. Most studies on longevity have been done in animals that live only a few weeks. Would the same set of genes that affect longevity in yeast, Caenorhabditis elegans, and Drosophila be revealed from a longevity screen involving animals that live much longer?
Bats have already been studied for their marked regenerative potential. Most mammals, including humans and mice, are poor at tissue regeneration, but bats are an exception. A common assay in bats for tissue regeneration is the generation of a large hole in the bat wing membrane. This hole is readily sealed without generation of scar tissue, a hallmark of regeneration. The enormous variance exhibited between mouse and bat, both in terms of morphology and biological function pertinent to human health, should provide readily discernible phenotypes for screening of bat chromosomal fragments to identify the causative elements giving rise to this variance.
We propose to use the Cre/loxP base site–specific recombination system to transfer defined bat chromosomal fragments to a chosen mouse docking site, each containing a defined small bat chromosomal fragment containing approximately 200 genes. Since the bat fragment is integrated into a mouse chromosome at a defined site it will be stably maintained through meiotic and mitotic cell divisions. The mice containing the defined bat chromosomal fragment will be screened for characteristics that distinguish mice from bats.
The bat project (MGWFACT) should yield a wealth of information that was previously unattainable. Almost 25 percent of mammalian species are bats, which have adapted through flight to an enormous range of habitats. Despite their small size and high rates of metabolism, they have extended life spans. Furthermore, this technology should be applicable to any mammal, each of which has a great deal to teach us, as each has adapted to a specific environmental niche. For example, there has been considerable interest in humanizing the mouse so that its pathology more closely reflects human disease. To date such efforts have been rather crude, one gene at a time. This technology provides a much more robust means for achieving such ends.
Grants from the National Institute of Child Health and Human Development, the National Institute of General Medical Sciences, and the National Institute of Mental Health provided partial support for these projects.
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